All vertebrates produce one or more different types of growth factors or growth hormones which are responsible for, among other effects, stimulating protein synthesis, cell division and growth, tissue formation, repair and/or maintenance, and storage or release of particular necessary nutrients into or out of specific cells. Such factors or hormones are often proteins or polypeptides they share the common feature of being manufactured and released by one type of cell, but exerting their ultimate effects on a different type of target cell. Among some of the better known growth mediators are nerve growth factor, epidermal growth factor, fibroblast growth factor, insulin-like growth factors (somatomedin) and growth hormone (somatotropin).
Each of these factors acts initially by binding to a receptor protein, which may be located either on the surface or in the cytoplasm of the particular factor's target cell. The receptor has a binding site which has a high affinity and specificity for the growth factor or hormone when the binding between factor and receptor occurs, a sequence of reactions is initiated which in some manner alters the functioning of the target cell. For example, it may cause the target cell to increase production and secretion of a particular protein, or alternately, it may signal the target cell to temporarily cease or decrease production of a certain protein.
Another example of a specific type of intercellular signalling compound is the peptide somatostatin, also referred to as somatotropin release-inhibiting factor (SRIF). This peptide is produced in a number of tissues, including the brain, gut, pancreas and adrenal cortex, and has wide-ranging effects throughout the body. As its name implies, SRIF inhibits the release of somatotropin from the anterior pituitary. However, other physiological effects of SRIF include inhibition of glucagon and insulin release from the pancreas, regulation of gut motility, and neurotransmission/neuromodulation function in the brain. In the latter capacity, SRIF can stimulate the release of neurotransmitters such as serotonin, dopamine and epinephrine. SRIF exists in two principle forms in mammalian tissues: SRIF-14, which is a tetradecapeptide, and SRIF-28, in which residues 15-28 are identical to SRIF-14. SRIF-28 also has an additional 14 amino acids at its amino terminus. The shorter peptide results from postranslational proteolytic cleavage of SRIF-28.
The intracellular aspect of the signalling action of somatostatin is mediated via receptors on the surface of target cells. It has been suggested that SRIF receptor subtypes exist in different cells and for differential interactions with specific signalling mechanisms. For example, radiological competition assays have shown that SRIF-14, SRIF-28 and other synthetic analogs can show vast differences in receptor binding affinity in brain, pancreas and pituitary cells (Srikant et al., Endocrinology 108:341-343, 1981; Tran et al., Science 228:492-495, 1985; Heiman et al., Neuroendocrinology 45:429-436, 1987; Amhardt et al., J. Clin. Invest. 80:1455-1460, 1987; Srikant et al., Nature 294:259-260, 1981). SRIF receptor subtypes are also suggested by the observation of differential effects of SRIF-14 and SRIF-28 on glucagon and insulin release in pancreas (Brown et al., Endocrinology 108:2391-2393, 1981) and on K.sup.+ channel activation in cultured cerebral cortical neurons (Wang et al., PNAS USA 86:9616-9620, 1989).
The activity of the SRIF receptor in mediating the SRIF biological effects seems to be intimately associated with pertussis toxin-sensitive GTP-binding regulatory proteins (hereinafter referred to as G proteins). It has been suggested (Jacobs et al., PNAS USA 80:3899-3902, 1983; Lewis et al. PNAS USA 83:9035-9039, 1985; Wang et al., PNAS USA 86:9616-9620, 1989) that SRIF receptors couple to cellular effector systems such as the adenylyl cyclase complex and to ion conductance channels by way of the G proteins. This association has been particularly well demonstrated in pituitary cells, wherein pertussis toxin blocked SRIF-mediated inhibitions of Ca.sup.2+ influx (Lewis et al., supra, Reisine et al., J. Pharmacol. Exp. Ther. 235:551-557, 1985) and adenylate cyclase (Reisine et al., J. Pharmacol Exp. Ther. 232:275-282, 1985), reduced agonist binding affinity of the SRIF receptor (Reisine, Supra), and blocked the SRIF-mediated inhibiting of adenylate cyclase and Ca.sup.2+ and decreased binding of [.sup.125 I]Tyr.sup.1 -SRIF by more than 95% (Kirk et al., Endocrinol. 114:1784-1790, 1984).
There has been evidence that direct immunoneutralization of SRIF enhances growth in primitive breeds of sheep (Spencer, Domestic Animal Endocrinology 3:55-68, 1985). However, this technique has had very limited success in commercial breeds of farm animals (Meats, Can. J. Anim. Sci. 70:1091-1097, 1990). This failure could be the result of compensatory overproduction of SRIF, variability in the immune response between animals or undesirable effects on other SRIF-dependent systems (i.e., lack of tissue specificity). However, the potentiation of growth hormone releasing factor effects in the rat model system by passive immunoneutralizatlon of SRIF (Wehrenberg et al., Endocrinology 114:1613-1616, 1984; Wehrenberg et al., Biochem. Biophys. Res. Comm. 109:562-567, 1982) suggests that neutralization of SRIF, either pharmacologically or immunologically, has great potential. One way in which the problems with the antisomatostatin approach might be overcome is the specific antagonism of the SRIF receptor via receptor-specific ligands or antireceptor antibodies. Use of either one of these techniques involves binding a non-SRIF material to the receptor, thereby blocking the receptor site and preventing the sending of endogenous SRIF. However, to date there has been little success in synthesizing peptide SRIF antagonists, and a random screening procedure would be useful in identifying compounds that inhibit SRIF activity. Moreover, although the immunological approach is becoming a means of animal growth regulation, the appropriate tools for application of these methods to SRIF have not yet been developed. In each of these approaches, the availability of a purified, well-characterized, tissue-specific receptor is essential to successful use of the methods.
Notwithstanding the need in art for isolation of SRIF receptors generally, and a pituitary receptor in particular, there has been little progress made in conclusively solubilizing and purifying well-defined SRIF receptors from any tissue type. Purification of pituitary receptors in general has been difficult because of the scarcity of tissue, as well as the problems involved in solubilizing the receptors in active form and developing an efficient purification method. SRIF receptors from various cell sources have been solubilized (He et al., Mol. Pharmacol. 37:614-621, 1990; Knuhtsen et al., J. Biol. Chem. 265:1129-1133, 1990; Reyl-Desmars et al., J. Biol. Chem. 264:18789-18795, 1989) in active, high affinity states in the detergent CHAPS. SRIF Receptor: [.sup.125 I] SRIF complexes have also been solubilized in CHAPS (Knuhtsen et al., Biochem. J. 254:641-647, 1988) and Zwittergent (Zeggari et al., Eur. J. Biochem. 164:667-673, 1987). At best, however, these complexes were stable enough to allow chromatographic separation of free from bound radio-ligand, and no further purification of receptor was reported.
One attempt to purify the solubilized SRIF receptor from rat brain has been reported (He et al., PNAS USA 86:1480-1484, 1989). The authors employed affinity chromatography on immobilized D-Trp-SRIF14. Eluates from the affinity column were not shown to have SRIF binding activity. Nonetheless, these investigators concluded that the SRIF receptor had been isolated. This conclusion was based on the ability to chemically cross-link a [.sup.125 I]-labelled SRIF analog to a 55-60,000 MW protein in the eluate, and comparison with a similar sized protein which was radiolabelled by chemical cross-linking in intact brain membranes (Thermos and Reisine, Mol. Pharmacol 33:370-377, 1988). The human gut SRIF receptor from HGT-1 cells was said to be purified by a monoclonal antibody with apparent specificity for the SRIF receptor (Reyl-Desmars, et al., J. Biol. Chem. 264:18789-18795, 1989). Eluates from the immunoaffinity column showed binding of [.sup.125 I]SRIF, and also contained a single narrow 90,000 MW band. However, the affinity column eluates showed greatly lowered SRIF binding affinity, making characterization of the purified receptor less reliable. Also, the sharply focused 90K band was unusual for a presumably glycosylated protein. Proteins of this type usually give a poorly focused "fuzzy" appearance. No further information has been published on any of these putative receptors; therefore, their identity as such remains uncertain.
The present invention now provides a substantially pure SRIF receptor protein, the identity of which is verified by a number of different criteria. The receptor protein, which is purified by a novel method, is isolated substantially free of other associated proteins, including G proteins, and as such is useful for sequencing, gene cloning, antibody production, and therapeutic purposes.